FEMS Yeast Research (2002) 279^289 www.fems-microbiology.org Molecular identi¢cation and genetic diversity within species of the genera Hanseniaspora and Kloeckera Neza Cadez a aYb , Peter Raspor a , Arthur W.A.M de Cock b , Teun Boekhout b , Maudy Th Smith bY * Biotechnical Faculty, Department of Food Science and Technology, University of Ljubljana, Jamnikarjeva 101, 1000 Ljubljana, Slovenia b Centraalbureau voor Schimmelcultures, Yeast Division, P.O Box 85167, 3508 AD Utrecht, Netherlands Received 24 April 2001 ; received in revised form 13 September 2001; accepted 27 September 2001 First published online 20 November 2001 Abstract Three molecular methods, RAPD-PCR analysis, electrophoretic karyotyping and RFLP of the PCR-amplified ITS regions (ITS1, ITS2 and the intervening 5.8S rDNA), were studied for accurate identification of Hanseniaspora and Kloeckera species as well as for determining inter- and intraspecific relationships of 74 strains isolated from different sources and/or geographically distinct regions Of these three methods, PCR-RFLP analysis of ITS regions with restriction enzymes DdeI and HinfI is proposed as a rapid identification method to discriminate unambiguously between all six Hanseniaspora species and the single non-ascospore-forming apiculate yeast species Kloeckera lindneri Electrophoretic karyotyping produced chromosomal profiles by which the seven species could be divided into four groups sharing similar karyotypes Although most of the 60 strains examined exhibited a common species-specific pattern, a different degree of chromosomal-length polymorphism and a variable number of chromosomal DNA fragments were observed within species Cluster analysis of the combined RAPD-PCR fingerprints obtained with one 10-mer primer, two microsatellite primers and one minisatellite primer generated clusters which with a few exceptions are in agreement with the groups as earlier recognized in DNA^DNA homology studies ß 2002 Federation of European Microbiological Societies Published by Elsevier Science B.V All rights reserved Keywords : Apiculate yeast; Identi¢cation ; PCR-RFLP analysis of rDNA ; Electrophoretic karyotyping ; RAPD-PCR analysis; Fingerprinting Introduction The ascomycetous yeast genus Hanseniaspora and its anamorph Kloeckera are morphologically characterized as apiculate yeasts with bipolar budding The species of the genera are frequently isolated from various natural sources such as soil, fruits and insects [1], as well as from fermented foods and beverages [2,3] As predominant inhabitants on the surface of grape berries and in starting wine fermentations, these genera have been intensively studied to determine their e¡ect on the quality of the ¢nal fermentation product Recently, it has been suggested that the presence of apiculate yeasts in the initial phases of wine fermentation contributes to a more complex and better aroma of the wine because of higher production of * Corresponding author Tel : +31 (30) 212 2666; Fax: +31 (30) 251 2097 E-mail address : smith@cbs.knaw.nl (M.T Smith) glycerol, esters and acetoin Strains of Hanseniaspora and Kloeckera are therefore potential candidates for mixed starter cultures [4^7] Several approaches have been applied to separate the species of Hanseniaspora and Kloeckera and to determine the relationships between teleomorph and anamorph species Besides physiological and morphological determinations [8^10], serology [11], proton magnetic resonance spectra of cell wall mannans [12], and DNA base composition [13] have been studied Currently, on the basis of DNA relatedness substantiated with physiological and morphological examinations, six teleomorph species with their anamorph counterparts and one anamorph species, Kloeckera lindneri, without a known teleomorphic state are accepted [14^16] The present classi¢cation was also con¢rmed by phylogenetic studies based on parts of large and small subunit ribosomal-DNA nucleotide sequences Sequence comparisons revealed that the genus Hanseniaspora is monophyletic and divided into two subgroups [17^ 20] The conventional identi¢cation key to discriminate between Hanseniaspora and Kloeckera species is based 1567-1356 / 02 / $22.00 ß 2002 Federation of European Microbiological Societies Published by Elsevier Science B.V All rights reserved PII: S - ( ) 0 - FEMSYR 1433 7-3-02 280 N Cadez et al / FEMS Yeast Research (2002) 279^289 on fermentation and/or assimilation of a few carbon sources and ability to grow at di¡erent temperatures The latter is the sole characteristic for di¡erentiating the closely related species Hanseniaspora osmophila and Hanseniaspora vineae or Hanseniaspora uvarum and Hanseniaspora guilliermondii [15] However, this characteristic can vary due to adaptation to di¡erent environments [21] To avoid doubtful identi¢cations or misidenti¢cations, genotypic methods which generate results independent of environmental conditions have been applied to food-borne strains, wine yeast strains and some type strains of Hanseniaspora and Kloeckera species [22,23] Esteve-Zarzoso et al [22] evaluated the use of restriction fragments length polymorphism (RFLP) of rDNA ampli¢ed by polymerase chain reaction (PCR) for the rapid identi¢cation of foodborne yeasts They found that discrimination among selected species of Hanseniaspora was possible using certain speci¢ed restriction enzymes Intraspeci¢c variation mostly of species important for winemaking such as H uvarum^ Kloeckera apiculata and H guilliermondii was studied by RAPD-PCR analysis [24], electrophoretic karyotyping [25,26] and AFLP ¢ngerprinting [27] In our study, we have used three molecular methods, (a) RAPD-PCR analysis, (b) electrophoretic karyotyping and (c) RFLP of the PCR-ampli¢ed ITS regions (ITS1, ITS2 and the intervening 5.8S rDNA), to examine the type strains of all currently accepted species along with other strains isolated from di¡erent sources and/or geographically distinct regions The species identity of these strains has been based on physiology and partly on DNA^DNA reassociations RAPD-PCR analysis has been used to evaluate the previously published statement [28] that high similarity in RAPD patterns correlates with high DNA homology Further, we have applied the RFLP analyses and karyotyping to evaluate their ability for accurate identi¢cation of all Hanseniaspora and Kloeckera species Moreover, we have determined inter- and intraspeci¢c relationships which were compared with relationships based on DNA^DNA homology studies [14] and sequencing analysis of rDNA [17,20] Materials and methods 2.1 Yeast strains The strains studied, their designations and origin, are listed in Table 2.2 Isolation of DNA for PCR assay DNA was isolated according to the method of Moller « et al [29] The DNA concentration was spectrophotometrically quanti¢ed and brought to a ¢nal value of 100 ng Wl31 2.3 RAPD-PCR analysis For a preliminary assay of RAPD-PCR analysis two strains of each species were selected We examined 19 decamer primers of arbitrary sequence from the OPA set (Operon Technologies Inc., Alameda, CA, USA), three microsatellite primers, (ATG)5 , (GTG)5 and (GTC)5 , and M13 core sequence (5P-GAGGGTGGCGGTTCT) For detailed analysis OPA-13 (5P-CAGCACCCAC) as 10-mer primer, (ATG)5 , (GTG)5 and M13 core sequence were selected Ampli¢cation reactions were performed in a ¢nal volume of 50 Wl containing 100 ng of genomic DNA, 10 mM Tris^HCl, 50 mM KCl, 1.5 mM MgCl2 , 0.001% gelatine, mM of each dNTP, 10 pM of primer and U of Taq DNA polymerase The thermal cycler was programmed for 40 cycles of at 94³C, at 60³C for primers M13 and (GTG)5 , at 48³C for (ATG)5 and at 36³C for the OPA primer set, followed by at 72³C PCR products were separated on 1.7% agarose gels in 1UTAE bu¡er chilled at 14³C To avoid ambiguous results, the ampli¢cation reactions of all 74 strains were processed simultaneously from one stock solution of premixed reagents in a single PCR assay as suggested by Messner et al [28] The RAPD-PCR pro¢les obtained with M13, (ATG)5 , (GTG)5 and OPA-13 of each strain were combined in a composite ¢ngerprint using GelCompar 3.1 (Applied Math, Kortrijk, Belgium) Similarities between combined ¢ngerprints were calculated using the Pearson product^ moment correlation coeÔcient (r) Cluster analysis of the pairwise values was generated using UPGMA algorithm 2.4 PFGE karyotyping Yeast chromosomes were isolated by a method described by Carle and Olson [30] as modi¢ed by Raspor et al [31] The chromosomal elements were separated in 1% agarose gels in 0.5UTBE bu¡er chilled at 12³C in a CHEF-DRII electrophoresis apparatus (Bio-Rad, Hercules, CA, USA) Electrophoresis was performed at 100 V for 36 h with a 200^300 s ramping switch interval and for 60 h with a 300^600 s ramping switch interval The electrophoresis for separation of H uvarum chromosomal fragments was prolonged and carried out at 100 V for 88 h with a 200^600 s ramping switch interval and then for 32 h at a 600^1200 s ramping switch interval The molecular sizes of the chromosomal bands ranging from 2800 to 1000 kb were calculated by comparison to a calibration curve based on Pichia canadensis (Hansenula wingei), those smaller than 1000 kb to Saccharomyces cerevisiae chromosomal DNA markers (Bio-Rad, Hercules, CA, USA) using the GelCompar 3.1 (Applied Math, Kortrijk, Belgium) computer program The inaccuracy of the sizes of the chromosomal elements in range from 300 kb to 1500 kb was 50 kb maximally FEMSYR 1433 7-3-02 N Cadez et al / FEMS Yeast Research (2002) 279^289 Table List of Hanseniaspora and Kloeckera strains studied Straina H guilliermondii CBS 465 CBS 95 CBS 466 CBS 1972 CBS 2567 CBS 2574 CBS 2591 CBS 4378 CBS 8733 NCAIM 741 (ZIM H occidentalis CBS 2592 CBS 280 CBS 282 CBS 283 CBS 2569 CBS 6782 H osmophila CBS 313 CBS 105 CBS 106 CBS 1999 CBS 2157 CBS 4266 CBS 6554 NCAIM 726 (ZIM H uvarum CBS 314 CBS 104 CBS 276 CBS 279 CBS 286 CBS 287 CBS 312 CBS 2570 CBS 2579 CBS 2580 CBS 2582 CBS 2583 CBS 2584 CBS 2585 CBS 2586 CBS 2587 CBS 2588 CBS 2589 CBS 5073 CBS 5074 CBS 5450 CBS 5914 CBS 5934 CBS 6617 CBS 8130 CBS 8734 CBS 8773 CBS 8774 CBS 8775 NCAIM 674 (ZIM NCAIM 725 (ZIM CCY 25-6-19 CCY 46-1-2 CCY 46-3-11 ZIM 1846 Statusb Origin of the strain T Infected nail, South Africa Fermenting bottled tomatoes, The Netherlands Dates Grape juice, Italy Grape must, Israel Grape juice, Italy Trachea of bee, France Caecum of baboon Opuntia megacantha, Hawaii, USA Orange juice concentrate, Georgia, USA T of Hanseniaspora meligeri ST of Hanseniaspora apuliensis ST of H guilliermondii T of Kloeckera apis 213, CBS 8772) T, T T of T of T of of Pseudosaccharomyces occidentalis Pseudosaccharomyces antillarum Pseudosaccharomyces javanicus Pseudosaccharomyces jensenii Soil, St Croix, West Indies Soil, Java Soil, Java Soil, Java Drosophila sp Orange juice, Italy T T T T K osmophila Pseudosaccharomyces magnus Pseudosaccharomyces corticis Pseudosaccharomyces santacruzensis Ripe Reisling grape, Germany Grape Bark of tree, Germany Soil, St Croix, West Indies Flower of Trifolium repens, Germany Cider, UK Patent (Takeda Chemicals Industries) Pineapple juice concentrate, Georgia, USA of of of of 212) T of Kloeckeraspora uvarum T of Pseudosaccharomyces apiculatus T of Kloeckera brevis T of Pseudosaccharomyces malaianus T of Pseudosaccharomyces muelleri T of Pseudosaccharomyces austriacus T of Pseudosaccharomyces germanicus T of Kloeckera lodderi AUT of K brevis 216) 211, CBS 8771) FEMSYR 1433 7-3-02 Muscatel grape, Crimea, Russia ? Soil, Germany Institute of Brewing, Japan Soil, Java Soil, Java Fermented cacao, Ghana Drosophila sp., Brazil Soil, Austria Soil, Germany Throat, The Netherlands Fermenting cucumber brine, USA ? Sour dough, Portugal Caterpillar Fruit must, Austria Tanning £uid, France Grape must, Italy Wine grape, Chile Apple grape, Chile Sea water, Florida, USA ? Cider, Illinois, USA Fruit of Musa sapientum Grapes, Italy Fruit of Sapindus sp., Hawaii, USA Flower from Schotia tree, South Africa Flower from Schotia tree, South Africa Flower from Schotia tree, South Africa Botanical garden pond, Hungary Spoiled grape punch, Georgia, USA Slovakia Slovakia Slovakia Grape must, Slovenia 281 282 N Cadez et al / FEMS Yeast Research (2002) 279^289 Table (continued) Straina NC-1 H valbyensis CBS 479 CBS 281 CBS 311 CBS 2590 CBS 6558 CBS 6618 NCAIM 330 (ZIM 229) NCAIM 642 (ZIM 224) H vineae CBS 2171 CBS 277 CBS 2568 CBS 6555 CBS 8031 K lindneri CBS 285 Statusb Origin of the strain Flower of Strelitzia sp., South Africa T T of Kloeckera japonica Soil, Germany Sap of tree, Japan Beer, Hungary Draught beer, England, UK Pulque, Mexico Tomato, Japan ? Cauli£ower, California, USA T of Kloeckera corticis T T of Pseudosaccharomyces africanus T of Hanseniaspora nodinigri Soil of vineyard, South Africa Soil, Algeria Drosophila persimilis Patent (Takeda Chemicals Industries) Black knot gall on Prunus virgin, Canada T of Pseudosaccharomyces lindneri Soil, Java a CBS, Centraalbureau voor Schimmelcultures, The Netherlands; ZIM, Culture Collection of Industrial Microorganisms, Slovenia; CCY, Culture Collection of Yeasts, Slovakia; NCAIM, National Collection of Agricultural and Industrial Microorganisms, Hungary b T, type strain; AUT, authentic strain; ST, syntype 2.5 PCR-RFLP analysis of rDNA The primers used for ampli¢cation of ITS regions were ITS1 (5P-TCCGTAGGTGAACCTGCGG) and ITS4 (5PTCCTCCGCTTATTGATATGC) as described by White et al [32] The ¢nal volume of the PCR reaction mixture was 50 Wl containing 100 ng of genomic DNA, 10 mM Tris^HCl, 50 mM KCl, 1.5 mM MgCl2 , 0.001% gelatine, mM of each dNTP, 50 pM of each of a pair of primers and U of Taq DNA polymerase (Promega, Madison, WI, USA) For ampli¢cation of ITS rDNA the PCR conditions were as follows : an initial denaturing step of at 94³C was followed by 35 cycles of 40 s at 94³C, 40 s at 56³C and 30 s at 72³C and terminated with a ¢nal extension step of at 72³C and cooling down to 4³C Restriction patterns of the PCR products were determined for each of the following 11 restriction enzymes: AluI, CfoI, DdeI, HaeIII, HinfI, HpaII, MspI, NdeII, Sau3A, ScrFI and TaqI (Roche, Mannheim, Germany) Digestions were prepared according to the instructions of the manufacturer The resulting fragments were separated on 3% agarose gels in 1UTAE bu¡er Ethidium bromide-stained gels were documented by Polaroid 665 photography under UV light or by GelDoc 2000 (BioRad, Hercules, CA, USA) In ITS nine di¡erent restriction groups were observed which showed a total number of 64 di¡erent fragments with the 11 enzymes used A binary matrix was generated manually by scoring absence (0) or presence (1) of each fragment for each group Further analyses were performed using NTSYS software package version 2.0 [33] Similarity values were calculated using the Dice coeÔcient, which is equal to two times the number of bands in common between two restriction patterns, divided by the sum of all bands Dendrograms were generated using an unweighted pair group method with arithmetic average (UPGMA) algorithm Results 3.1 Growth at 34³C and 37³C According to Smith [15] the sibling species H vineae and H osmophila can be distinguished by the presence or absence of growth at 34³C, respectively, while the sibling species H uvarum and H guilliermondii can be discriminated by the absence or presence of growth at 37³C, respectively In order to evaluate these characteristics all strains of these four species were re-examined for growth at the aforementioned temperatures None of the H osmophila strains grew at 34³C as expected; however, two strains of H vineae, CBS 277 and CBS 2568, also failed to grow at this temperature All strains of H uvarum failed to grow at 37³C as expected; however, two strains of H guilliermondii, CBS 1972 and CBS 2567, also failed to grow at 37³C 3.2 RAPD-PCR analysis Among nineteen 10-mer primers and four microsatellite primers tested, the primers OPA-03, OPA-13, OPA-18 and (ATG)5 , (GTG)5 , and M13 core sequence yielded useful patterns to allow veri¢cation of the identity of strains These primers, except OPA-03 and OPA-18, were used in further studies FEMSYR 1433 7-3-02 N Cadez et al / FEMS Yeast Research (2002) 279^289 283 Fig RAPD ¢ngerprints of Hanseniaspora^Kloeckera strains generated with Opa-13 primer M, SmartLadder 200 bp (Eurogentec) The RAPD-PCR patterns of Hanseniaspora^Kloeckera using primer OPA-13 are shown in Fig Fig depicts the dendrogram derived from the combined RAPD-PCR ¢ngerprints after cluster analysis At the similarity level of 40% we could recognize eight clusters Generally, strains of the same species clustered together with a few exceptions Two strains of Hanseniaspora occidentalis, CBS 2569 and CBS 6782 (Fig 2, marked with arrows), did not cluster with the main group (cluster 6) Five strains of H uvarum (cluster 8) clustered at the similarity level of 20% far apart from the main group (cluster 1) which contained the type of this species Unpublished preliminary DNA homology studies showed this cluster to be di¡erent from H uvarum To settle the ¢nal taxonomic status of this cluster, further studies are needed, and, therefore, they were excluded from the rest of this study The single strain of K lindneri clustered among the isolates of Hanseniaspora valbyensis (cluster 4) showing a similarity of 49% to CBS 2590 3.3 Karyotyping In Fig and Table 2, only the CHEF karyotypes and estimated sizes of chromosomal DNA bands of the type strains of Hanseniaspora and Kloeckera species are presented These chromosomal pro¢les can be divided into four groups: group I contains the species H occidentalis, H vineae and H osmophila; group II H uvarum and H guilliermondii, and groups III and IV comprise H valbyensis and K lindneri, respectively Most of the examined strains showed a species-speci¢c pattern; however, chromosomal-length polymorphism (CLP) occurred and the number of chromosomal DNA bands was variable within the species (Fig 4) Three out of six strains of H occidentalis, CBS 2592T , CBS 2569 and CBS 6782 (Fig 4a), showed a similar banding pattern, with six chromosomal fragments ranging in size from 2600 kb to 900 kb, that di¡ered from the karyotypes of H vineae (Fig 4b) in a resolved third and fourth chromosomal fragment from the top The average size of the genome was ca 11.3 Mb The karyotypes of the three other strains of H occidentalis isolated from Java (Indonesia) were highly variable The karyotype of CBS 280 consisted of an additional chromosome of 1100 kb (Fig 4a, marked with an arrow) and it lacked the third chromosomal fragment Strain CBS 282 showed a pattern similar to that of the type strain CBS 2592 but two additional bands of 1300 kb and 1000 kb were present (Fig 4a, FEMSYR 1433 7-3-02 284 N Cadez et al / FEMS Yeast Research (2002) 279^289 Fig UPGMA cluster analysis of 74 digitized combined RAPD-PCR ¢ngerprints of Hanseniaspora^Kloeckera strains The distance between strains was calculated using the Pearson correlation coeÔcient (% r) FEMSYR 1433 7-3-02 N Cadez et al / FEMS Yeast Research (2002) 279^289 285 doubling of the smallest chromosomal fragments (e.g Fig 4d, CBS 8130, marked with an arrow), as well as in the size and number of the uppermost fragments (e.g Fig 4d, CBS 286, marked with arrows) Strain CBS 2586 exhibited the most divergent karyotype with the largest chromosomal fragment of ca 2.8 Mb and a total genome size of approx 15 Mb The karyotypes of H guilliermondii (Fig 4e) were similar to those of H uvarum (Fig 4d), with CLP occurring among the largest and the smallest chromosomal DNA fragments Strains of H valbyensis (Fig 4f) were found to have a di¡erent chromosomal pattern Seven to nine chromosomal DNA bands were resolved with sizes ranging from 0.75 to 2.6 Mb The average genome of this species is ca 11.7 Mb The intraspeci¢c CLP also occurs in this species Fig Electrophoretic karyotypes of Hanseniaspora^Kloeckera type strains after CHEF electrophoresis M1, chromosomal DNA of P canadensis YB-4662-VIA as size marker; M2, chromosomal DNA of S cerevisiae YNN295 as size marker (both Bio-Rad) 3.4 PCR-RFLP analysis of rDNA ITS regions were ampli¢ed separately from genomic DNA of the type strains of Hanseniaspora and Kloeckera species The ampli¢ed ITS regions were approximately 720 bp long, without any size variation between the strains on 1% agarose gel The preliminary PCR-RFLP analysis of the ITS regions with 11 restriction enzymes performed on the type strains of Hanseniaspora and Kloeckera revealed that MspI had no recognition site in the ITS regions and that Sau3A, NdeII and HpaII did not reveal any polymorphism Results obtained by the remaining seven restriction enzymes are presented in Table Of these seven enzymes, DdeI was suitable to di¡erentiate the types of all Hanseniaspora^Kloeckera species (Fig 5a) except H valbyensis and K lindneri, which could be di¡erentiated by HinfI (Fig 5b) or HaeIII (Table 3) To examine intraspeci¢c polymorphisms within the Hanseniaspora species, three enzymes, HaeIII, HinfI, and DdeI, were examined in more detail All strains of Hanseniaspora species exhibited restriction pro¢les identical to those of the type strain of the species with the exception marked with arrows) CBS 283 (Fig 4a) exhibited a signi¢cantly di¡erent pattern, similar to K lindneri CBS 285 (Fig 3), isolated also from soil in Java The chromosomal DNA of CBS 283 (Fig 4a) in the uppermost part of the gel remained unresolved whereas the remaining two bands occurred as doublets at ca 2200 kb and 1700 kb The karyotype of strains of H vineae (Fig 4b) contained ¢ve chromosomal DNA bands ranging from 2500 to 930 kb The estimated genome size varied between and 13 Mb Two strains, CBS 2568 and CBS 6555, contained additional faint DNA bands of 1600 and 2100 kb, respectively (Fig 4b marked with arrows) A species-speci¢c karyotype pattern of H uvarum (Fig 4d) consisted of six to nine chromosomal fragments, ranging in size from 2200 to 600 kb Doublet bands occurred at ca 1100 and 1000 kb and the average genome size is an estimated 9.6 Mb The most apparent di¡erences among the karyotypes of H uvarum were found in migration and Table Estimation of chromosome sizes of type strains of Hanseniaspora and Kloeckera species Type strain Group I H occidentalis H vineae H osmophila Group II H uvarum H guilliermondii Group III H valbyensis Group IV K lindneri Chromosome sizes (kb) CBS 2592 CBS 2171 CBS 313 2620 CBS 314 CBS 465 2400 2470 2400 2060 2340 2300 1840 1840 1810 1500 1430 1330 900 920 830 690 2180 2160 2110 1980 1610 1700 1430 1470 1080 1150 1040 1100 670 830 CBS 479 2580 2340 2010 1780 1640 1420 1170 CBS 285 2440 2100 1950 1600 1550 790 FEMSYR 1433 7-3-02 750 286 N Cadez et al / FEMS Yeast Research (2002) 279^289 Fig Electrophoretic karyotypes of strains H occidentalis (a), H vineae (b), H osmophila (c), H uvarum (d), H guilliermondii (e) and H valbyensis (f) M1 , chromosomal DNA of P canadensis YB-4662-VIA as size marker; M2 , chromosomal DNA of S cerevisiae YNN295 as size marker (both BioRad) The data sets from the ITS spacer digests were used to calculate similarity coeÔcients and to construct a dendrogram with NTSYS-pc The topology of the ITS-RFLP dendrogram (Fig 6) revealed four clusters of species with the similarity level ranging from 65% for the species H vineae and H osmophila to 95% for the sibling species H uvarum and H guilliermondii of H occidentalis strains Restriction enzyme HinfI divided the species into three groups: group I contained the type strain CBS 2592, CBS 280 and CBS 283, group II CBS 282 and group III CBS 6782 and CBS 2569 (Fig 5c) These subgroups were further examined with the other enzymes Only TaqI and AluI separated group II or group III from group I, respectively (Table 3) Table Restriction fragment patterns of ITS regions of Hanseniaspora and Kloeckera generated by seven restriction enzymes (A^G)a Enzyme Species H occ H vin I ScrFI CfoI AluI HaeIII DdeI TaqI HinfI II A1 B1 C1 D1 E1 F1 G1 A1 B1 C1 D1 E1 F2 G2 A1 B1 C2 D1 E1 F1 G3 H osm H uvar H guill H valb K lind A1 B2 C3 D2 E2 F3 G4 A1 B2 C3 H3 E3 F4 G4 A1 B3 C4 H4 E4 F5 G5 A1 B3 C4 H4 E5 F5 G5 A2 B4 C5 H4 E6 F6 G6 A2 B4 C5 H5 E6 F6 G7 III Within each enzyme di¡erent patterns were numbered successively, starting with number for the ¢rst pattern Identical numbers within an enzyme indicate identical patterns a MspI has no recognition site in the ITS regions; Sau3A, NdeII and HpaII not reveal polymorphism FEMSYR 1433 7-3-02 N Cadez et al / FEMS Yeast Research (2002) 279^289 287 Fig PCR-RFLP analysis of ITS region of Hanseniaspora^Kloeckera type strains listed in Table with restriction enzymes DdeI (a) and HinfI (b,c) M1 , SmartLadder 200 bp (Eurogentec); M2 , 100-bp ladder (Gibco BRL) Hocc, H occidentalis; Hvin, H vineae; Hosm, H osmophila; Huva, H uvarum; Hguill, H guilliermondii ; Hval, H valbyensis; Kl, K lindneri Discussion A polyphasic approach, which integrates phenotypic, genotypic and phylogenetic information, provides reliable information about relationships among species and strains This study presents a contribution to the characterization of intraspeci¢c variation and interspeci¢c relationships of yeasts belonging to the genera Hanseniaspora and Kloeckera We found that PCR-RFLP analysis of ITS regions with two restriction enzymes allowed discrimination of all species : DdeI restriction patterns were speciesspeci¢c for all species examined, except H valbyensis and K lindneri Discrimination between the latter two was possible using HinfI Moreover, HinfI divided H occidentalis into three subgroups The development of a molecular identi¢cation key was provoked by inconsistencies in identi¢cation results reported by Vaughan-Martini et al [26] Testing the growth ability at 34³C and 37³C, being key characteristics in the current identi¢cation key [15,16], we con¢rmed their ¢ndings : strains which were found to be conspeci¢c on the basis of high DNA homology were variable with regard to growth at 34³C or 37³C De Morais et al [21] suggested that variations in ability to grow at higher temperatures may be a consequence of adaptation to the environment Two strains of H guilliermondii, CBS 1972 and CBS 2567, however, failed to grow at 37³C, although they were both isolated from warmer climates (Italy and Israel, respectively) than some other strains of this species (Table 1) The cluster analysis of the combined RAPD-PCR ¢ngerprints revealed groups that agreed with those obtained by DNA^DNA homology studies [14] Each cluster represented a currently accepted species in the genus Hanseniaspora, and one separate cluster of ¢ve strains represented a group of strains physiologically undistinguishable from H uvarum The intraspeci¢c similarity values ranged from 40 to 68%, which is quite low compared to the values reported for P membranifaciens [34] However, strains of the latter species were all isolated from the same substrate, whereas the strains of Hanseniaspora were isolated from Fig UPGMA cluster analysis of Hanseniaspora^Kloeckera strains listed in Table based on ITS restriction patterns FEMSYR 1433 7-3-02 288 N Cadez et al / FEMS Yeast Research (2002) 279^289 di¡erent sources Species boundaries agreed with correlation values of below 38% The RAPD-PCR analysis did not re£ect phylogenetic relationships between the species, not even the relationship between the closest related species H vineae and H osmophila sharing 40% DNA^DNA homology [14] Therefore, the method is only useful for revealing the relationships among strains within species of Hanseniaspora due to its high resolution capacity Based on the results of electrophoretic karyotyping, the genera Hanseniaspora and Kloeckera can be divided into four subgroups sharing similar karyotypes The phylogenetically closely related species H vineae^H osmophila and H uvarum^H guilliermondii [17,20] have similar karyotypes These species are also diÔcult to discriminate by conventional criteria currently employed in yeast taxonomy [15] On the other hand, the species H valbyensis and its closest related anamorph species K lindneri di¡er markedly by their chromosomal DNA pattern and physiologically they can also be di¡erentiated by their maximal growth temperature [16] The observed CLP of strains of H uvarum from diverse geographical origin is comparable with that of H uvarum strains isolated from Malvasia grape juice [35] and therefore does not re£ect the presence of several distinct populations but merely indicates the rapid karyotypic changes which may occur within populations [36] De Barros Lopos et al [27] observed by AFLP genotypic analysis that most strains of H uvarum are genetically rather uniform and they correlated the close genetic relatedness with the in£uence of humans on their dispersal and consequently the lack of genetically distinct populations This hypothesis is con¢rmed by uniformity of our RAPD ¢ngerprints (Figs and 2) of H uvarum strains, which were isolated mostly from man-made environments Although the estimated genome size by PFGE is hampered by the possible presence of doublet or triplet chromosomes and the occurrence of similar-sized heterologous chromosomes, the average estimated genome sizes of 9.6 Mb of H uvarum strains in our study is in accordance with previous estimates of 9.9^10 Mb [25] Identi¢cation of Hanseniaspora isolates by PCR-RFLP of ITS regions has been applied recently by Esteve-Zarzoso et al [22] albeit for a restricted number of species In another study, Dlauchy et al [23] proposed the use of AluI for the di¡erentiation of these closely related species However, we found no AluI restriction polymorphisms in the ITS regions between H vineae and H osmophila nor between H uvarum and H guilliermondii The dichotomy of the genus Hanseniaspora supported by phylogenetic studies [17,20] was not con¢rmed with the ITSRFLP dendrogram However, the ITS-RFLP dendrogram showed a high relatedness (95% similarity) between H uvarum and H guilliermondii, which was also con¢rmed by the low number of nucleotide substitutions in the D1/D2 domain of the 26S rDNA [20] On the other hand, a similarity value of only 65% between H vineae and H osmophila did not correlate with rDNA sequencing [17,20] and DNA homology data [14] The latter study showed that H vineae and H osmophila were more closely related species sharing 38^60% DNA^DNA homology values, while the closely related H uvarum and H guilliermondii shared only 11^29% DNA^DNA homology High intraspeci¢c variation of the strains of H occidentalis was revealed by all three methods used The highest variation was found in the electrophoretic karyotypes Groupings observed in the PCR-RFLP of rDNA were less distinct than those in the karyotypes The genotypic methods used in our study to characterize strains of Hanseniaspora and Kloeckera were directed towards di¡erent aspects of the genome, such as the ribosomal gene, the mini-, microsatellite and random sequences, and the analysis of the chromosomal make-up All three methods con¢rmed the relationships within species of the genus Hanseniaspora and the status of the anamorph species K lindneri In particular restriction analysis of rDNA is a reliable and rapid method for the identi¢cation of Hanseniaspora^Kloeckera isolates Acknowledgements This study was supported by a FEMS fellowship granted to N.C References [1] Barnett, J.A., Payne, R.W and Yarrow, D (2000) Yeasts: Characteristics and IdentiÂcation, 1139 pp Cambridge University Press, Cambridge ă [2] Deak, T and Beuchat, L.R (1996) Handbook of Food Spoilage Yeasts CRC Press, Boca Raton, FL [3] Heard, G.M and 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chromosomal fragment from the top The average size of the genome was ca 11.3 Mb The karyotypes of the three other strains of. .. occurred and the number of chromosomal DNA bands was variable within the species (Fig 4) Three out of six strains of H occidentalis, CBS 2592T , CBS 2569 and CBS 6782 (Fig 4a), showed a similar banding